Neuronal organelle transport has been intensively studied using in vitro systems, and numerous candidates for the motors that drive the movement of organelles have been identified by primary sequence. However, from the biochemical to the behavioral level, we still know very little about how organelle transport is controlled and coordinated in intact cells. The first major aim of this study is to elucidate the nature and regulatory function of kinesin phosphorylation. Kinesin is phosphorylated in vivo, but the effects on its motor function and organelle binding remain uncertain, due to contradictory results obtained from in vitro versus in vivo studies. In addition, the physiologically-relevant kinase(s) are unknown. These questions will be addressed using metabolic labeling neurons, phosphopeptide sequencing, site-directed mutagenesis, transfection and a cell fractionation assay. In order to under-stand how the regulation of motor proteins gives rise to coordinated organelle transport, it is necessary both to test mechanistic hypotheses and to continue to quantitatively determine the full range of regulated organelle traffic that actually occurs in cells. Thus, the second major aim is to determine how organelle transport is modulated to meet specific physiological needs of the cell. Experiments will focus on the regulation of mitochondrial motility by the NGF/TrkA signaling pathway, and on the coordination of mitochondrial movement with metabolic state. This direction will be pursued by focal NGF stimulation, quantification of organelle behavior, and determination of the metabolic state of mitochondria in live neurons via quantitative microscopy. The third major aim will be to explore a recently recognized element of axonal transport, the movement and deposition of mRNA within the axon. Experiments will employ in situ hybridization and transfection of primary cultured neurons with labeled products of an axonal cDNA library to determine the rate, mechanism, and function of axonal mRNA transport. Because axonal transport provides a metabolic link between the periphery of neurons and the major site of synthesis in the cell body, it is essential for the development and maintenance of all nerves; thus, the insight into its mechanism gained by the proposed study can be expected to contribute to our understanding of the development, defects, and diseases of the nervous system.